ABSTRACT Our laboratory aims to understand in detail how iron transport influences erythropoiesis and how the erythron communicates iron requirements to the liver. Iron for erythropoiesis is transported by the iron transporter transferrin (Tf) and iron uptake by erythroid precursors requires Tf-Fe binding to transferrin receptor 1 (TfR1). In turn, erythroid precursors regulate iron metabolism in part by secreting factors, such as the recently identified erythroferrone (ERFE), which suppresses the hormone hepcidin, a key inhibitor of dietary iron absorption, recycling from senescent RBCs, and mobilization from iron stores. ERFE is increased in models of both stress (i.e. phlebotomized wild type mice) and ineffective erythropoiesis (i.e. ?-thalassemic mice). Furthermore, erythroid precursors express iron and heme export proteins, the function of which remains unclear. We have shown that exogenous iron-free Tf ameliorates ineffective erythropoiesis in ?-thalassemic mice (Li et al., Nat Med, 2010), resulting in decreased ERFE, increased hepcidin, and relative systemic iron deficiency. We hypothesize that the beneficial effect of exogenous Tf in ?-thalassemic mice is a consequence of more than frank iron restriction. Specifically, our preliminary data suggests that Tf functions via an effect on TfR1, influencing enucleation through effects on membrane TfR1 as well as iron metabolism indirectly by decreasing Erfe expression and directly by decreasing soluble TfR1. Furthermore, surprisingly, hepcidin expression is not suppressed in TfR1+/- mice, despite relative iron deficient erythropoiesis and increased ERFE expression. Thus, we hypothesize that the beneficial effect of exogenous Tf on ineffective erythropoiesis is a consequence of reduced TfR1 expression or altered TfR1 trafficking from erythroid precursors. Here we propose to define in detail how changes in Tf concentration, iron uptake and efflux, and TfR1 trafficking in the erythron influence erythropoiesis and erythroid hepcidin regulation. In the proposed three specific aims, we will 1) assess the regulatory role of Tf and TfR1 in iron restricted and ineffective erythropoiesis; 2) examine the significance of TfR1 as a regulator of hepcidin; and 3) elucidate the effect of Tf and TfR1 on iron efflux from erythroid precursors and hepatocytes. Iron transport for erythropoiesis and erythroid regulation of iron metabolism are central tenets in recovery during stress erythropoiesis and are dysregulated in ineffective erythropoiesis. Thus, successful completion of these studies elucidating the role of Tf and TfR1 in these pathways is of great interest to the hematology field, provides insight into the pathophysiology of human diseases in which erythropoiesis is disturbed, extends current knowledge in iron metabolism, and adds new paradigms for further exploring erythroid regulation of hepcidin. Lastly, the successful completion of these studies interrogating mechanisms involved in stress and ineffective erythropoiesis (responsive to PAS-13-031) will facilitate the development of clinical trials using Tf in patients with ?-thalassemia and possibly other iron-loading anemias.